Friedreich's ataxia (FRDA) is the most frequent inherited ataxia found in individuals of Indo-European and North African descent. In majority of cases, the disease is caused by inheritance of two mutant alleles carrying expanded GAA/TTC tracks that inhibit FRDA gene expression. Besides FRDA, there are thousands of other GAA loci in the human genome that have potential for the expansions. The molecular mechanisms of GAA expansions and other associated instabilities are poorly understood. The goal of this research is the elucidation in detail of how expanded Friedreich's ataxia GAA trinucleotide repeats lead to chromosomal fragility and subsequent chromosomal rearrangements, as well as the effect of fragility on size variations in yeast, Saccharomyces cerevisiae. This is the first study of GAA-mediated fragility and its consequences on genome integrity in a eukaryotic model organism. The Specific Aim 1 is to determine the molecular mechanisms of chromosomal fragility induced by expanded tracks of GAA triplet repeats. The hypothesis that length- and orientation-dependent formation of triplex DNA by GAA repeats leads to replication arrest, subsequent attack of the secondary structure by the mismatch repair complex and generation of double-strand break will be tested. It will be achieved by assessing fragility in chromosomal arm loss and gene amplification assays, and by examining replication arrest and DSB intermediates in wild type and mutant strains. The Specific Aim 2 is to characterize the structural organization of gross chromosomal rearrangements resulting from GAA-mediated breakage. The rearranged chromosomes will be analyzed using contour-clamp homogeneous electric-field gel electrophoresis, Southern analysis, comparative genomic hybridization on microarrays and molecular combing followed by fluorescent in-situ hybridization. The sequence composition of the rearrangement breakpoints will be also determined. The Specific aim 3 is to establish the role of the fragility in the generation of repeat-size variations. The frequencies of deletions and expansions during mitotic and meiotic divisions will be estimated by using PCR and Southern analyses. The hypothesis that fragility can stimulate long size variations will be tested by comparing the frequencies of repeat size alterations between wild type and strains that are deficient in double-strand break formation or repair. The results of this research will provide the insights into mechanisms of somatic and germline instabilities of GAA repeats observed at the FRDA locus (in Friedreich's ataxia patients) and at other GAA- containing loci in the human genome. In addition, this study will contribute to the understanding of instabilities associated with other types of disease-causing sequence motifs. PUBLIV HEALTH RELEVANCE: This research aims to elucidate the molecular mechanisms underlying GAA trinucleotide repeat instability. This study is important for understanding the etiology and pathology of the repeat-associated disorders as well as for determining the susceptibility of the carriers with expanded tracks to the tumorigenic chromosome aberrations.